A tumor glycome laboratory team with multi-disciplinary expertise in glycomics, proteomics, informatics, and cancer markers is proposing to implement a new paradigm in the use of glycan biomarkers for early detection of cancer. We will focus on a primary problem in cancer of significant unmet need, namely identification of glycans and glycoproteins that are applicable to the early detection of aggressive breast cancer defined on the basis of estrogen and progesterone (ER-.PR-) receptor negative tumor status. The proposal includes two discovery aims and a validation aim. Specific aim one is focused on the analysis and identification of glycoproteins in plasma and tissue to detect differences between breast cancer subjects and controls. Specific aim two will identify and characterize the glycan changes associated with these glycoproteins;to obtain enough sample, tissue tumor containing and tumor free tissue will used. For glycoproteins of interest, the number of glycosylation sites, the attachment point of the glycan to the polypeptide backbone and the accurate mass and fragmentation pattern of individual glycopeptides in the LT-FTMS and LTMS with both CID and ETD will be determined. Additionally glycans will be cleaved and their structure determined after permethylation by MALDI-TOF analysis of the resulting glycan HPLC profiles. Candidate glycoprotein markers resulting from these specific aims will be prioritized based on elevated levels in both breast tumor tissue and plasma and occurrence of altered glycosylation. The translational aspect of this study is achieved in specific aim three by the development of a high throughput and sensitive glycan assay platform (GAP) which will be used to pre-validate markers in a large sample set and thus facilitate subsequent clinical studies. The project will benefit from substantial glycomic, glycoproteomics and clinical resources available to the applicant group.